The haematopoietic system is a complex hierarchy of cells of different mature cell lineages. These include cells of the immune system that offer protection from pathogens, cells that carry oxygen through the body and cells involved in wound healing. All these mature cells are derived from a pool of haematopoietic stem cells (HSCs) that are capable of self-renewal and differentiation into any blood cell lineage.
As HSCs have the ability to replenish the entire haematopoietic system, they may be used for transplantations, for example following haematotoxic insults such as radiotherapy or chemotherapy, or for the replacement of leukaemic cells.
Hematopoietic cell transplantation (HCT) is a curative therapy for several inherited and acquired disorders. However, allogeneic HCT is limited by the poor availability of matched donors, and the mortality associated with the allogeneic procedure which is mostly related to graft-versus-host disease (GvHD) and infectious complications provoked by the profound and long-lasting state of immune dysfunction.
Gene therapy approaches based on the transplantation of genetically modified autologous HSCs offer potentially improved safety and efficacy over allogeneic HCT. They are particularly relevant for patients lacking a matched donor.
The concept of stem cell gene therapy is based on the genetic modification of a relatively small number of stem cells. These persist long-term in the body by undergoing self-renewal, and generate large numbers of genetically “corrected” progeny. This ensures a continuous supply of corrected cells for the rest of the patient's lifetime. HSCs are particularly attractive targets for gene therapy since their genetic modification will be passed to all the blood cell lineages as they differentiate. Furthermore, HSCs can be easily and safely obtained, for example from bone marrow, mobilised peripheral blood and umbilical cord blood.
Efficient long-term gene modification of HSCs and their progeny requires a technology which permits stable integration of the corrective DNA into the genome, without affecting HSC function.
Long-term benefit requires the transplantation of a sufficiently high number of modified HSCs, which can repopulate the conditioned bone marrow, giving rise to corrected blood cells of all hematopoietic lineages. Autologous HSCs therefore make the transplant procedure available to all patients, avoid the immunological compatibility problems leading to GvHD and allow minimally immunosuppressive conditioning regimens thus drastically reducing infectious complications.
Lentiviral-based HSC gene therapy trials have demonstrated their therapeutic potential in curing genetic diseases. However, difficulties remain with the methods employed for the genetic modification of HSCs.
Current HSC gene therapy protocols (e.g. Cartier N et al. Science 2009; 326:818-823; Cavazzana-Calvo M et al. Nature 2010; 467:318-322; Biffi A et al. Science 2013; 341:1233158; Aiuti A et al. Science. 2013; 341:1233151) use a 2-4 day ex vivo culture during the HSC genetic modification process. Longer culture times typically yield higher transduction levels. However, ex vivo culture negatively impacts on HSC function and this negative effect clearly correlates with the duration of culture (Guenechea G et al. Blood 1999; 93:1097-1105; Xu R et al. Transfusion 2001; 41:213-218; Mazurier F et al. Blood 2004; 103:545-552; Ahmed et al. Blood 2004; 103:2079-2087; Glimm H et al. Exp. Hematol. 2005; 33:20-25; Kallinikou K et al. Br. J. Haematol. 2012; 158:778-787). Although some progress has been made towards improving the ex vivo expansion of HSCs (Zhang C C et al. Blood 2008; 111:3415-3423; Boitano A E et al. Science 2010; 329:1345-1348; Delaney C et al. Nat. Med. 2010; 16:232-236; Himburg H A et al. Nat. Med. 2010; 16:475-482; Csaszar E et al. Cell Stem Cell 2012; 10:218-229; Walasek M A et al. Ann. N. Y. Acad. Sci. 2012; 1266:138-150), the resulting protocols present several challenges for clinical translation, give variable and often poorly reproducible results, and still need to be proven in relevant clinical settings. Consequently ex vivo culture in the context of HSC gene therapy should be kept as short as possible.
Accordingly, there is a need for devising improved protocols that allow efficient genetic modification of haematopoietic stem cells while minimising culture time. Furthermore, the improved protocols need to be suitable for clinical use.